The identification of three-dimensional structural relationships in intact biological tissue specimens using micro-optical techniques conventionally entails problems due to the fact that optical transparency of such tissue samples is often largely lacking. This relates in particular to the investigation of structural relationships between neurons in nerve tissue or brain tissue. Conventional methods provide, for this purpose, for the sensing of microscopic serial sections in the form of digital images, from which a reconstruction of the three-dimensional relationships is then performed. This is, however, at least very laborious and in some cases error-prone.
In light of this, a recent publication (K. Chung et al., Structural and molecular interrogation of intact biological systems, Nature 497, 332-337, 2013) proposes a method for increasing the optical transparency of regions of a corresponding tissue sample. As explained therein, the lack of optical transparency is attributable substantially to lipid bilayers. Incoming light is scattered at interfaces formed thereby. The aforementioned publication therefore proposes the nondestructive removal of such lipid bilayers, i.e. a removal such that the remaining tissue sample is structurally maintained to the greatest possible extent.
For this the tissue sample is impregnated, for example, with hydrogel monomers (e.g. acrylamide and bisacrylamide), formaldehyde, and thermoreactive initiators. The formaldehyde on the one hand ensures crosslinking of the tissue, and on the other hand the hydrogel monomers couple covalently to biomolecules such as proteins and nucleic acids. Polymerization of the correspondingly coupled monomers is then thermally initiated to yield a hydrogel network. The aforesaid biomolecules are incorporated into the three-dimensional hydrogel thereby created, and thus stabilized.
Molecules having no couplable functional groups, in particular lipids of the lipid bilayers, remain unbound and can therefore be extracted from the structure that has been created. An ionic extraction method is usefully utilized for this, since micro-optical investigation, e.g. using fluorescence microscopy, is not thereby influenced.
For extraction of the lipids it is possible to use, for example, sodium dodecyl sulfate (SDS) micelles in aqueous solvents, which because of their net negative charge in neutral to alkaline conditions migrate in an applied electric field and can thereby be driven through the correspondingly prepared tissue sample.
Reference may be made to the aforementioned publication for further details of a corresponding method. It should be emphasized, however, that the present invention not only can be utilized in the specific method variant disclosed therein, but also can be used in other methods in which a gradual increase in the optical transparency of regions of a tissue sample can be observed at least in certain method steps.
Such methods are conventionally carried out entirely manually, i.e. the outcome of a corresponding method is monitored visually by an experimenter. Such methods are thus, in practice, work-intensive and poorly reproducible. The latter circumstance proves to be disadvantageous especially in the context of medical research projects, since its consequence is that standardized sample preparation is not possible.
The invention intends to provide a remedy here, and to simplify methods for increasing the optical transparency of regions of tissue samples and especially to improve them in terms of increased reproducibility.